title: Acetylcholine leads to signal transducer and activator of transcription 1 (STAT-1) mediated oxidative/nitrosative stress in human bronchial epithelial cell line.
abstract: The induction of nitric oxide synthase (iNOS) expression via the signal transducer and activator of transcription 1 (STAT-1) is involved in the mechanism of oxidative/nitrosative stress. We investigated whether acetylcholine (ACh) generates oxidative/nitrosative stress in bronchial epithelial cells during airway inflammation of COPD and evaluated the effects of Tiotropium, a once-daily antimuscarinic drug, and Olodaterol, a long-acting β2-agonist on these mechanisms. Human bronchial epithelial cells (16-HBE) were stimulated (4h, 37°C) with induced sputum supernatants (ISSs) from healthy controls (HC) (n=10), healthy smokers (HS) (n=10) or COPD patients (n=10), as well as with ACh (from 1μM to 100μM). The activation of STAT-1 pathway (STAT-1Ser727 and STAT-1Tyr701) and iNOS was evaluated in the cell lysates by Western blot analysis as well as nitrotyrosine levels by ELISA, while reactive oxygen species (ROS) were evaluated by flow cytometry. Finally, the effect of Tiotropium (Spiriva®) (100nM), alone or in combination with Olodaterol (1nM), was tested in this model. ISSs from COPD patients significantly increased the phosphorylation of STAT-1Ser727 and STAT-1Tyr701, iNOS and ROS/Nitrotyrosine when compared with ISSs from HC or HS subjects in 16-HBE cells. Furthermore, synthetic ACh increased all these parameters in stimulated 16HBE when compared with untreated cells. Tiotropium and Olodaterol reduced the oxidative/nitrosative stress generated by ACh and ISSs. We concluded that ACh mediated the oxidative/nitrosative stress involving the STAT-1 pathway activation in human bronchial epithelial cells during COPD. β2-Long acting and antimuscarinic drugs, normally used in the treatment of COPD as bronchodilator, might be able to control these cellular events.
authors.affiliation: Unit: "Ex vivo/In Vitro Models to study the Immunopathology and the Pharmacology of airway diseases", Institute of Biomedicine and Molecular Immunology (IBIM), Italian National Research Council (CNR), Palermo, Italy. Electronic address: profita@ibim.cnr.it.
authors.forename: Mirella
authors.initials: M
authors.lastname: Profita
authors.forename: Giusy Daniela
authors.initials: GD
authors.lastname: Albano
authors.forename: Angela Marina
authors.initials: AM
authors.lastname: Montalbano
authors.forename: Caterina
authors.initials: C
authors.lastname: Di Sano
authors.forename: Giulia
authors.initials: G
authors.lastname: Anzalone
authors.forename: Rosalia
authors.initials: R
authors.lastname: Gagliardo
authors.forename: Loredana
authors.initials: L
authors.lastname: Riccobono
authors.forename: Anna
authors.initials: A
authors.lastname: Bonanno
authors.forename: Liboria
authors.initials: L
authors.lastname: Siena
authors.forename: Michael Paul
authors.initials: MP
authors.lastname: Pieper
authors.forename: Mark
authors.initials: M
authors.lastname: Gjomarkaj
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D015153:Blotting, Western; D001980:Bronchi / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D029424:Pulmonary Disease, Chronic Obstructive / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D034741:RNA, Small Interfering / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D018679:Cholinergic Agonists; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D050794:STAT1 Transcription Factor; C002744:3-nitrotyrosine; D014443:Tyrosine; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D000109:Acetylcholine
keywords: Acetylcholine; Epithelial cell; Oxidative/nitrosative stress; STAT-1